Trained Immunity Generated by the Recombinant Zoster Vaccine

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes, natural killer (NK) cells, and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte, DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied.


Introduction
Herpes zoster (HZ) is a severe disease caused by the reactivation of latent varicella-zoster virus (VZV).Asymptomatic VZV reactivations are not uncommon, but viral replication is generally controlled by the host immune response.Clinical manifestations of HZ ensue when the immune system fails to contain the viral spread.HZ is most frequent in people with decreased cell-mediated immunity (CMI), including those with congenital immunode ciencies, AIDS, iatrogenic immunosuppression, and older adults with immunosenescence [1][2][3][4] .Moreover, the risk of HZ is not increased by isolated antibody de ciency, nor is it mitigated by providing antibody supplementation.Thus, protection against HZ is deemed to be primarily mediated by CMI.Additional evidence links the incidence of HZ to a decrease in VZV-speci c Th1 responses 5 .However, the exact mechanisms that limit viral spread during VZV reactivation have not been de ned.In addition to adaptive immunity, protection against HZ may involve innate cell-mediated immunity [6][7][8] .Innate immune cells are rapidly deployed as the rst line of defense against pathogens.Moreover, innate immune cells are critical for the development of antigen-speci c adaptive CMI and for establishing feedback mechanisms that allow adaptive CMI to boost innate immunity [9][10][11][12][13][14][15] .Recent evidence revealed that innate immune cells develop memory-like responses, also known as trained immunity 11,[15][16][17][18][19][20][21][22][23][24][25][26] .Unlike conventional T and B cells, innate immune cells either lack antigen-speci c receptors or have a limited capacity for recombination.Instead, the development of memory in innate immune cells relies on epigenetic modi cations and clonal expansions 26,27 .The best studied viral infection controlled by trained immunity is cytomegalovirus (CMV), which, like VZV, is a member of the Herpesviridae family that is de ned by a life cycle consisting of acute infection, followed by latency and reactivation.CMV-speci c adaptive NK cell clones, which recognize CMV peptides in the context of HLA class I E, have high in vitro cytolytic capacity against CMV-infected cells and confer in vivo protection in a murine CMV infection model and in human transplant recipients [27][28][29][30][31] .These adaptive NK cells have well-characterized epigenetic modi cations underlying their CMV speci city 27,32,33 .VZV-speci c adaptive NK cells have also been described 34 .
Another set of innate immune cells with CMV-speci c activity are the Vδ2 − and Vγ9 − γδ T cells [35][36][37] .These cells undergo limited T-cell receptor (TCR) rearrangement, which confers CMV speci city and the ability to control CMV reactivations [35][36][37][38] .Monocytes and dendritic cells (DCs) can also develop trained immunity 26,39 .The role of monocytes in the control of viral infection has been insu ciently studied, but monocytes in the context of M. tuberculosis infection develop trained immunity that confers protection against active disease 40 .Monocyte immunologic memory is maintained through epigenetic modi cations of hematopoietic stem and progenitor cells 41,42 .
Vaccines are available to protect against many infections, including HZ, but people at the highest risk of developing these infections tend to have poor responses to vaccines.This is exempli ed by the Zoster Vaccine Live (ZVL), which confers 70% protection against HZ in adults 50 to 59 years of age, 64% in adults aged 60-69 years, 41% in adults aged 70-79 years and no protection in adults aged ≥ 80 years 43-45   .Moreover, the protection conferred by ZVL completely wanes 10 years after vaccination.In contrast, a recombinant zoster vaccine (RZV) -containing the VZV glycoprotein E (gE) combined with the potent adjuvant ASO1 B -changed this paradigm.RZV achieved 3-year e cacy > 90% and 10-year e cacy > 70% against zoster in healthy adults ≥ 50 years of age.E cacy and persistence were minimally affected by the age of the vaccinee [46][47][48] .We previously showed that RZV generated higher levels of gE-and VZV-speci c CD4 + Th1 memory, CD8 + cytotoxic lymphocytes, and antibodies [49][50][51][52] than ZVL.However, adaptive T cell immune responses measured after administration of live attenuated viral vaccines typically require a week to reach their peak 53 .Thus, it is likely that innate immune responses are essential for initial control of infection with most pathogens.The ability of RZV to generate trained immunity has not been studied, but non-VZV vaccines, including live-attenuated, adjuvanted and mRNA vaccines, were shown to induce trained immunity 21,[54][55][56][57][58][59] .
In the current study, we reveal VZV-speci c innate immune responses in peripheral blood mononuclear cells (PBMC) after RZV administration, but not after ZVL, and then demonstrate that RZV generates durable gE-speci c homologous and heterologous trained immunity in monocytes, NK, and DC that involves epigenetic modi cations.

Demographic characteristics of the study population
This study used samples from 10 ZVL and 25 RZV recipients enrolled in a randomized, double-blind study comparing the safety and immunogenicity of the two vaccines (Supplemental Fig. 1; Supplemental Table 1).Participants who contributed samples to this study had mean (range) ages of 67 (50-79)  years.Of the 35 total participants, 18 were females and 33 were White Non-Hispanics without appreciable differences between the vaccine groups.

The frequency of activated innate and adaptive immune cells in peripheral blood increases only after RZV administration
To determine the innate immune response to zoster vaccines, we measured the activation of monocytes, DC, NK and γδ T cells in peripheral blood up to 30 days after each dose of vaccine in 10 ZVL and 10 RZV recipients.For reference, we measured activation of conventional CD4 + and CD8 + T cells and B cells representing adaptive immunity.Activation of B cells, monocytes, and DC was measured by expression of PDL1 and of the other lymphocytes by dual expression of HLA DR and CD38 (Gating strategy shown in Supplemental Fig. 2).We observed transient responses to vaccination in peripheral blood both in innate cells and conventional T cells but with diverging kinetics.CD4 + effector memory T cells (Tem) signi cantly increased after each dose of RZV with peaks on Days 7 and 67 (p ≤ 0.01; Fig. 1) and showed a trend increase between Days 7 and 67 (p = 0.098).ZVL recipients showed increases in activated CD4 + Tem on Day 7 that did not quite reach statistical signi cance (p = 0.06).In contrast, activated innate immune cells and CD8 + Tem increased in peripheral blood on Days 1 and/or 61 post-RZV compared to pre-vaccination levels.Activated γδ T cells reached signi cantly higher levels both at Days 1 and 61 post-RZV (p ≤ 0.02) compared to pre-vaccination without a signi cant difference between the two time points post-vaccination, while activated DC had a marginal increase on Day 1 (p = 0.09) and signi cant increase on Day 61 (p = 0.01) compared to pre-vaccination and signi cant increase from Day 1 to 61 (p = 0.02).The numbers of activated CD8 + Tems, monocytes and NK cells did not increase in the peripheral blood on Day 1 but did signi cantly increase on Day 61 compared to pre-vaccination or Day 1 levels.We did not detect increased activation in the peripheral blood of B cells in either vaccine group (not depicted) or in the innate cells or CD8 + Tems in the 10 ZVL recipients (Fig. 1).
Innate immune cells may be activated by cytokines generally secreted by conventional T cells-a process known as bystander activation 60 .However, differences in the kinetics of activation between CD4 + Tems and innate immune cells argue against bystander activation.Correlation analyses revealed signi cant associations only between activated CD4 + Tems or activated total CD4 + T cells and activated γδ T cells on Days 1 and 61 (rho ≥ 0.9 and p ≤ 0.001 by Spearman correlation analysis).The activation of other innate immune cells did not correlate with either CD4 + or CD8 + T-cell activation.

Parallel development of trained and adaptive immune responses after RZV administration
To test the hypothesis that RZV generates trained immunity, we stimulated ex vivo PBMCs from 10 RZV recipients with VZV-gE overlapping peptides (gE pp) or recombinant VZV-gE (rgE) and medium unstimulated controls and from 10 ZVL recipients with medium and replication competent VZV.The stimulation was performed on PBMCs obtained from all study visits in the rst year after vaccination.
Activation was measured by the expression of PDL1 on B cells, DCs, and monocytes and by the coexpression of CD25 and CD137 on NK cells, γδ, and conventional T cells under antigen-stimulated conditions after the subtraction of unstimulated controls (the gating strategy is shown in Supplemental Fig. 3).Compared with prevaccination, RZV recipients showed activation of B cells, DCs, monocytes, NKs, and conventional T cells on Day 7, which returned to prevaccination levels on Day 30, increased again on Day 67 (7 days after the 2nd dose of vaccine) and persisted above prevaccination on levels on Days 90 and 365 (Fig. 2).Activated γδ T cells also increased above prevaccination levels in response to antigenic stimulation on Days 7, 67 and 90 but returned to prevaccination levels on Day 365.ZVL recipients showed increased activation of B cells and conventional CD4 + T cells up to Day 365, while DC and conventional CD8 + T cells increased only at Days 7 and 30 (Supplemental Fig. 4).
Correlation analysis of the proportions of CD4 + T cells activated by VZV-gE ex vivo in RZV recipients with activated B cells, CD8 + T cells, DCs, monocytes, NK cells and γδ T cells revealed signi cant associations between CD4 + T cells and CD8 + T cells, γδ, and NK cells on Days 0 and 7 (Supplemental Table 2).Correlations between CD4 + and CD8 + T-cell activation were also noted on Days 90 and 365 (Supplemental Table 2).No other signi cant associations were noted, such that bystander activation of B cells, DCs, monocytes, γδ T cells and NK cells seemed unlikely after the 2nd dose of the vaccine.

Homologous trained immunity of DCs, monocytes and NK cells persists for several years after RZV administration
To investigate the persistence of innate immune responses generated by RZV, we stimulated ex vivo PBMCs from 14 RZV recipients at 0, 3, 12, 48, and 60 months postvaccination with rgE and a medium negative control (see methods).We found that DC and monocyte responses to rgE persisted above prevaccination levels for ≥ 5 years and that NK cells persisted for 4 years (Fig. 3).In contrast, following the administration of RZV, we could not observe responses to the nonspeci c inducers R848 or rhIL2 used at suboptimal doses to identify vaccine-induced increased responses (Supplemental Fig. 5).

Puri ed monocytes and NK cells from RZV recipients exhibit homologous and heterologous trained immunity
Monocytes and NK cells were isolated from PBMCs obtained on Days 0 and 90 from 10 RZV recipients by negative depletion using magnetic beads (see Methods section).We then combined monocytes and NK cells from the same donor at a ratio of 2:1, roughly replicating their ratios in PBMCs.The resulting cocultures had < 5% T-cell contamination.Cocultures were stimulated with rgE, CMV lysate, herpes simplex virus (HSV) lysate and medium control overnight, and activation was measured by the expression of PDL1 on monocytes and the coexpression of CD69 and CD137 on NK cells (Fig. 4).There was an increase in activation from pre-to postvaccination, both in NKs and monocytes, in response to all antigens.Notably, puri ed monocytes in isolation did not respond to ex vivo rgE stimulation, suggesting that cross talk between monocytes and NK cells is essential for activation.

Epigenetic modi cations associated with RZV administration
Monocytes and other immune cells develop immunologic memory through epigenetic modi cations 26 .To determine whether the monocyte memory responses to RZV were associated with epigenetic modi cations, we performed bulk ATAC-seq on monocytes puri ed by sorting from PBMCs obtained before and 90 days after the rst dose of vaccine in 8 RZV recipients.We found 16 loci with signi cantly modi ed chromatin accessibility de ned by ≥ 2-fold differences in gene expression from pre-to postvaccination and an FDR-adjusted p value < 0.1 (Fig. 5A).Among the genes that may play a role in the immune response, tgfβ1, dnam2, and arap1 exhibited decreased expression, and mgll and efna5 exhibited increased expression.
ATAC-seq analysis of puri ed NK cells from 10 participants revealed differential expression of 13 genes from pre-to postimmunization (Fig. 5B).However, we did not identify any genes associated with immune responses.

TGFβ1 modulates ex vivo monocyte responses to rgE in RZV recipients
We hypothesized that downregulation of the TGFβ1 pathway contributed to the trained immunity developed by monocytes in response to RZV administration.To test this hypothesis, we treated ex vivo puri ed monocyte and NK cell cocultures from 10 RZV recipients with rhTGFβ1 or with the compound LY3200882 (LY), which prevents the binding of TGFβ to its receptor and subsequent downstream signal transduction.Treatment of cells subjected to prevaccination with LY increased their activation in response to rgE stimulation to levels comparable to those of cells subjected to rgE-induced activation on Day 90 postvaccination (Fig. 6).Conversely, treatment of cells collected on Day 90 with rhTGFβ1 decreased rgE-induced activation to levels similar to those observed before vaccination.NK cells only partially responded to ex vivo modulation of TGFβ1 activity (Supplemental Fig. 6), suggesting that additional mechanisms may contribute to the development of trained immunity in NK cells.

Discussion
The homologous trained immunity generated by vaccines may play an important role in protection against infections.For example, homologous trained immunity resulting from BCG or Ad26-SIV vectored vaccines was shown to contribute to protection against tuberculosis and simian immunode ciency viral infections, respectively 21,54 .Trained innate immune cells may also play a role in protection against HZ 34 .Here, we show that the highly e cacious RZV generates strong innate immune responses.The activation of NK cells, γδ T cells, monocytes and DCs in the blood was detected one day after the administration of each dose of RZV.Moreover, after the 2nd dose of RZV, gE-stimulated ex vivo activation of NK cells, DCs and monocytes remained persistently greater than that before vaccination for at least 5 years, suggesting the development of trained immunity.Using their rapid response capability, innate immune cells can provide the initial response to the replication of reactivated VZV, ensuring that the reactivation of latent VZV does not progress to HZ.
The monocyte, DC and NK responses to VZV-gE ex vivo did not correlate with CD4 + T-cell responses, suggesting the development of trained immunity rather than a bystander stimulation effect.Moreover, we con rmed the development of gE-speci c trained immunity in puri ed monocyte and NK cocultures.Notably, isolated monocytes were not differentially activated by rgE before or after vaccination.Only after the addition of puri ed NK cells to the puri ed monocyte cultures was the presence of trained immunity revealed.This observation suggested that cross talk between monocytes and other cells is essential for trained immunity generated by RZV.NK cells express CD28 61,62 , which can bind to CD80 or CD86 on the monocyte cell membrane, while monocytes express HLA Class I E 63 , which is recognized by NK cells.We propose that rgE or gE pp mediate the binding of these receptors and ligands, forming an immunologic synapse between monocytes and NK cells, which is essential for RZV-generated trained immunity.
We were also able to demonstrate monocyte and NK heterologous trained immunity against other herpesviruses, CMV and HSV, which is supported by clinical observations associating RZV administration with protection against COVID-19 64 .However, we did not observe increased heterologous responses to R848 stimulation of TLR7 and TLR8 or rhIL2.The absence of trained immunity to R848 may be explained by the divergence of intracellular pathways activated by antigens and TLR agonists.Binding of TLRs 7 and 8 elicits downstream activation of interferon regulatory factors and NFκB 65,66 .In contrast, peptide binding to MHC activates Src kinases, and subsequent tyrosine phosphorylation is the main intracellular downstream signaling mechanism 67 .Moreover, T-cell recognition of the peptides presented in the context of MHC on APCs creates an immunologic synapse where CD80, CD86, and CD40 APC receptors bind to their corresponding T-cell ligands, triggering additional activation signals in the APC 68,69 distinct from those generated by R848 binding to TLRs.We propose that cell imprinting resulting from RZV administration modulates the downstream signaling pathways triggered by peptide binding to MHC and/or costimulatory receptors on APCs but not the pathways activated by TLR7/8 stimulation or rhIL2 treatment.
Innate immune cells acquire memory through epigenetic imprinting.In the case of short-lived myeloid cells, such as monocytes and macrophages, chromatin modi cations of hematopoietic stem and progenitor cells ensure the persistence of newly acquired characteristics 54,56 .For example, epigenetic features underlying trained immunity against tuberculosis and SARS-CoV-2 infection were shown to persist for 3 and 6 months after BCG administration and COVID-19 infection, respectively.Our investigation of the monocyte epigenome of RZV recipients revealed decreased accessibility of tgfβ1, dnm2 and arap1 and increased accessibility of efna5 and mgll after vaccination.
TGFβ is a regulatory cytokine that plays a key role in the differentiation of regulatory T cells and other cell subsets.We and others have shown that TGFβ inhibits the activation and proliferation of effector T cells and the functionality of NK cells [70][71][72] .TGFβ also increases the differentiation of monocytes into myeloidderived suppressor cells (MDSCs), which inhibit T-cell functionality, while neutralization or inhibition of TGFβ intracellular downstream signaling decreases differentiation into MDSCs in favor of differentiation into antigen-presenting DCs 73 .Thus, downregulation of TGFβ1 expression in monocytes after vaccination could explain the observed increase in monocyte function.Moreover, the addition of rhTGFβ1 to rgE-stimulated monocyte and NK cocultures signi cantly decreased monocyte and NK activation, while blocking TGFβ1 had the opposite effect.These ndings support the role of TGFβ1 downregulation in establishing monocyte trained immunity.
The other genes downregulated by RZV in monocytes are involved in endocytosis and cell receptor activity [74][75][76] , whereas the upregulated genes play a role in cell adhesion, extravasation, and migration 77 and in lipid metabolism and synthesis of arachidonic acid, the precursor of prostaglandins and other in ammatory mediators 78,79 .Modi cations in the expression of these genes may also contribute to protective immune responses against VZV.
Although NK cells also develop epigenetic modi cations after vaccination, the affected genes are not known to contribute to the immune response.CMV adaptive NK cells were previously shown to undergo epigenetic modi cations through DNA methylation 32 , which was not investigated in our study.Future analyses of the effect of RZV on the DNA methylation of NK cells are planned.
DC also showed evidence of homologous trained immunity in bulk PBMC cultures stimulated with rgE.Although the bulk cultures contained gE-speci c conventional T cells, we found that DC activation did not correlate with T-cell activation, suggesting that DCs were not exclusively activated by adaptive T cells.We did not pursue homologous or heterologous trained immunity in isolated DCs because they represent a very small cell population, and we did not have enough PBMCs to create isolated DCs or DC and NK cocultures, for example, to prove the development of DC trained immunity.Additional genomic assays, including multiome analyses, are planned to elucidate this aspect of DC functionality.
We observed only short-lived memory-like responses in γδ T cells after RZV.Both RZV and ZVL are associated with increased activation of CD4 + T cells and B cells, which are known to develop immunologic memory through receptor rearrangement.CD8 + T-cell activation after vaccination was observed only in RZV recipients.
Our study had limitations.Since there were no study visits between Days 1 and 7 post-vaccinations, we cannot rule out that additional increases in circulating activated innate and/or adaptive immune cells occurred between Days 1 and 7 in RZV and/or ZVL recipients.It is also possible that the relatively small number of samples tested in this proof-of-concept study might have limited our ability to detect signi cant increases in the innate immune responses of ZVL recipients.Moreover, prevaccination responses to VZV were greater than those to gE, making it more challenging to demonstrate postvaccination increases in response to VZV than to gE.This observation, coupled with the fact that gEspeci c responses after ZVL administration are very low 50 , limits our evaluation of increases in trained immunity in ZVL recipients.
In conclusion, we showed that RZV induces trained immunity in addition to adaptive immunity, revealing a novel attribute of this vaccine.The discovery of gE-speci c trained immunity elicited by RZV offers an opportunity to examine its role in protection against HZ, a disease for which a speci c immune correlate with protection has not yet been identi ed.

STAR Methods
Study design.This study (NCT02114333) was approved by the Colorado Multiple Institutional Review Board.All participants provided signed informed consent.The study enrolled 160 participants in good health except for those with treated chronic illnesses typical of the age of the vaccinees.All had prior varicella or resided in the USA for at least 30 years, and subsequent antibody testing by gp ELISA con rmed the presence of VZV-speci c antibodies in all participants 51 ; none had prior HZ.The exclusion criteria from the study were immune suppression and recent blood products or other vaccines.The participants were divided into two age groups: ≥50-59 years (n = 46) or ≥ 70-85 years (n = 115).The older group included 70 people who received ZVL ≥ 5 years before enrollment and 44 who did not.Participants in each group were randomized at enrollment to receive one dose of ZVL followed by placebo 60 days later or RZV in two doses separated by 60 days (Supplemental Fig. 1).Of the 160 enrollees, 159 were vaccinated.The current study used viably cryopreserved PBMCs from blood samples collected before and up to 5 years after vaccination from participants who did not receive prior ZVL.Flow cytometry assays.PBMCs were cryopreserved for viability within 8 h of collection and stored at ≤-150°C until use.For phenotypic characterization of innate and adaptive immune cells in peripheral blood (the gating strategy is shown in Supplemental Fig. 2), PBMCs were thawed, counted, and promptly stained with two different panels.Both staining panels included an initial PBS wash and viability staining with Zombie Yellow Fixability dye (BioLegend).For the APC and B-cell panels, the cells were subsequently washed with phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA; Sigma; A9576-50ML) and incubated at room temperature with Human TruStain FcX (BioLegend).Next, the cells were washed and stained for the surface markers CD14 Ax488, CD83 PE, CD141 PE-Dazzle, CD123 PerCPCy5.5,CD56 PE-Cy7, CD1c Ax700, HLA-DR APCcy7, PDL1 BV421 (BioLegend), CD3 PE-Cy7, CD19 APC (BD Biosciences), and True-Stain Monocyte Blocker (BioLegend).
Homologous and heterologous immunity of separated monocytes and NK cells.

Statistical analysis.
ATACseq analysis: fastq les were uploaded to the Galaxy platform 80 for analysis.FastQC was used to investigate read quality, and Cutadapt was used to cut out the adapter sequences.Reads were then mapped to the human reference genome (hg38) using Bowtie2.Uninformative reads were ltered out based on read mapping quality (< 30 on the phred quality scale), proper pairing, mitochondrial reads, and duplicate reads.The insert size distribution was then checked to verify the expected nucleosomal pattern.Files were converted from BAM to BED format, and peak calling was performed using MACS2.
The bedgraph output les from the peak calling were then converted to BigWig format for easier visualization of the genome.DiffBind was used to produce a normalized read count matrix, and the matrix was read into R 81 for differential chromatin accessibility analysis using DESeq2 82 .FDR was used to adjust for multiple comparisons, and modi cations were considered signi cant if the p value was < 0.1.The signi cantly modi ed genes were annotated using the biomaRt package 83, and Path ndR 84 was used for pathway analysis.
Flow cytometry data analysis: For the cell activation and persistence experiments, analysis of variance (ANOVA) with FDR adjustment was used to determine differences between timepoints using Prism 10.1.1 for MacOS software (GraphPad).Puri ed monocyte and NK culture experiments were analyzed using the Wilcoxon matched-pairs signed rank test to identify differences in paired data before and after vaccination.ANOVA for repeated measures was used to test differences between TGFβ1 treatments, and analysis was performed using Prism software as described above.Author contributions:

KEY RESOURCES
MJJ performed ow cytometry and molecular assays, participated in data analysis and manuscript preparation; DG and TV participated in statistical analysis; MJL lead the parent study and participated in Innate and adaptive immune cell activation in response to ex vivo antigenic stimulation in RZV recipients.
Data were derived from 10 participants who received 2 doses of RZV at enrollment and 60 later.

Figures
Figures

TABLE IFNg
AW receives grants from GlaxoSmithKline and Merck and personal fees from GlaxoSmithKline; MJL receives research grants and personal fees from GlaxoSmithKline and Curevo Vaccines.All other authors report no COI.